Selective lysing of cells using ultrasound

ABSTRACT

Described are embodiments that employ ultrasonic energy to selectively lyse larger adipose cells in a suspension containing adipose cells of different sizes resulting in a suspension in which the only viable cells are the small adipose cells and stem cells. Embodiments provide for generating an acoustic standing wave field of sufficient intensity and proper geometry, that high shear stress is induced on the cell membranes of cells larger than a predetermined size. The remaining small adipose cells can be physically separated from the suspension after the suspension is subjected to the acoustic standing wave field.

I. CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Patent Application Ser. No.61/266,867, filed Dec. 4, 2009 entitled “SELECTIVE LYSING OF CELLS USINGULTRASOUND,” the contents of which are hereby incorporated by referencein their entirety as if set forth herein in full.

II. FIELD OF THE INVENTION

Embodiments of the present invention relate to methods and devices foruse in the selective “lysing,” i.e., destroying, of cells usingultrasound. In particular, the embodiments relate to the selectivetermination of larger, generally older, adipose cells in a mixture ofadipose cells of various sizes and maturities such as found in anextract from animal, e.g., human, tissues.

III. BACKGROUND OF THE INVENTION

When fat cells are extracted from the body, typically in a liposuctionprocedure, they encompass a range of sizes, from less than 25 microns indiameter, to more than 100 microns in diameter. The smaller cells aretypically pre-adipocytes, while the larger cells are fully matureadipocytes. Many of the extracted adipose cells are “alive,” i.e., theysurvive separation from the surrounding tissue and the extractionprocess. Information suggests that when the extraction process isperformed using ultrasonic assisted lipoplasty (UAL) (such as thatperformed using the VASER® UAL system commercially available from SoundSurgical Technologies LLC in Louisville, Colo.), a greater portion ofthe extracted cells are alive and can survive for a limited period oftime post-extraction. The extracted cells are of interest in severalapplications.

In the practice of medicine, it is sometimes desired to re-inject theextracted adipose cells into the same patient for purposes of filling oraugmenting body locations, e.g., plastic surgery, body sculpting orenhancement. To optimize that process it is helpful to re-inject onlylive cells. Further, it would be preferable to inject only the smallercells, preferably the pre-adipocytes. This is because the pre-adipocytesmore readily adapt to the re-implantation process, and also stimulatethe production of the vascular structure necessary for successfulgrafting, through the expression of vascular growth factors. The moremature cells, on the other hand, do not generally express the vasculargrowth factors, and are more likely to undergo necrosis afterre-implantation. It is therefore desirable to separate the mature fatcells from the liposuction aspirate before re-implantation. Further, itmay be desired to destroy the mature fat cells, so that the only viablecells remaining are of the smaller, more active type. While an exactcut-off size which is optimal for re-insertion has not been clinicallyestablished, generally, it would be desirable to lyse those cells largerthan about 50 microns. However this may be considered on apatient-by-patient basis, for instance, depending upon the total amountof fat available for re-implantation, the amount required to bere-implanted in order to produce the desired result, the generalcondition of the patient's cells, etc.

Secondly, it has now been found that stem cells can be derived fromadipose cells, a discovery having significant implications for medicalresearch regarding both humans and animals. (See, for example, U.S. Pat.No. 6,777,231 for “Adipose-Derived Stem Cells and Lattices,” issued onAug. 17, 2004.) Young adipose cells are prime candidates for thederivation of stem cells. Stem cells are generally in the range of 15-25micron in diameter.

Stem cells can be used to regenerate human or other animal tissue. Onceisolated, stem cells can be reintroduced into a human or other animal toregenerate tissues, in vitro. Because stem cells may be reintroducedinto a human body, processing performed to isolate the stem cells mustbe performed in a sterile environment.

Thus, it would be desirable to be able to isolate the younger, smallercells from adipose tissue extracted from humans and other animals andeasily maintain a sterile environment for the stem cells to be safelyre-injected into a patient.

It is further desirable that this selection, separation, and/ordestruction process be accomplished in a rapid manner, as the timebetween “aspiration” (i.e., removal of tissue from the body via suction)and re-implantation or storage for research should be kept to a minimum.

Prior efforts in this area have involved centrifugation, which does notprecisely segregate cells of different sizes. Successful centrifugationalso requires a precise drawing off of the centrifuged material, whichrequires additional handling. Filtering of the aspirate is alsopossible, although this can also be a time consuming process, and canalso cause damage to the cells of interest as they pass through thefilter material. Also, since cells are often aggregated in the aspirateand not singulated, it is likely that a cluster of the desired, smallercells would be filtered out. For example, a cluster of more than a few50 micron diameter cells would be filtered out by a filter with a 150micron mesh, which defeats the purpose of the entire filtering process.The use of filtering and/or centrifugation also requires the use ofadditional devices that must be sterilized to avoid contaminating thestem cells.

Thus, neither centrifugation nor filtering has completely solved theproblem of isolating smaller, younger adipose cells. Centrifugation doesstratify the aspirate material, with smaller, denser cells at thebottom, and larger, less dense cells at the top of the centrifuge tube.However, the stratification is imprecise; it can be affected byclustering of the cells; and it can cause damage to the desired cells byboth additional handling and the stresses of centrifugation.

Similarly filtering the aspirate is also imprecise in that the tendencyof cells to be in clusters affects the filtering process.

Accordingly, a need still exists for an improved method of isolatingyounger adipose cells from an aspirate mixture and maintaining thesterility of the aspirate mixture.

IV. SUMMARY OF THE INVENTION

The summary is provided to introduce aspects of some embodiments of thepresent invention in a simplified form, and is not intended to identifykey or essential elements of the claimed invention, nor is it intendedto limit the scope of the claims.

Embodiments of the present invention employ ultrasonic energy toselectively lyse larger adipose cells in a suspension containing adiposecells of different sizes resulting in a suspension in which the onlyviable cells are the small adipose cells. The embodiments also providefor easily maintaining the sterility of the suspension during lysing byusing a cartridge as the chamber for holding the suspension when it isbeing lysed. The cartridge, in some embodiments, is used only a singletime, which reduces the risk of introducing contaminants into thesuspension. As used herein “lysis” refers to the destruction of cells, aprocess that frequently involves disruption of the cell membrane. Thus,the lysed cells spill their internal lipids into the suspension. As aresult, the remaining small adipose cells can be physically separatedfrom the suspension.

As used herein, “adipose cells,” “fat cells,” or “adipocyte” refers toany of various cells found in animal tissue that are specialized for thestorage of fat.

Acoustic standing waves have been used to move and sort particles bothin air and in liquids. In general, these systems have operated in adifferent way from embodiments of the present invention and haveresulted in acoustic separation, but not lysing.

In designing prior art systems, the selection of the acoustic standingwave field spacing and therefore the selection of frequency and chambersize, are dictated by fluidics issues, not necessarily cell size.Specifically, these prior systems were designed to provide as muchphysical separation of cells as possible, so that cells of differentcharacteristics that are influenced by acoustics (e.g., compressibilityand density) are as far from each other in the flow stream as possible.This simplified the downstream task of bifurcating the flow and havingeach flow stream have only one cell type. The design of the systemparameters, i.e. acoustic standing wave distance, operating frequency,flow rate, dwell time, and chamber size, did not take into considerationthe size of the cell nor the forces required to create lysing in the waythat is presented here.

The prior art attempted to have all the cells of a specific type or someacoustically differentiable characteristic in a single zone, and all thecells of a different characteristic in another zone, so that the twocell types could be easily separated by separation of the flow streamscontaining the different zones.

In contrast, embodiments of the invention described herein do not useultrasonic energy to cause physical separation of cells with differentacoustic attributes. Instead, the design rules of the presentembodiments are intended to lyse cells above a particular size therebyenabling the subsequent separation of the lysed cell remnants from theunlysed cells without reference to “zones,” i.e., flow separationdistances. Thus, for example, the embodiment of the present inventionillustrated in FIGS. 3 and 4, has cells of different characteristics (inthis case, different sizes) all intermixed both before and after theapplication of ultrasonic energy. To accomplish this result embodimentsof the present invention employ standing wave nodal distances which aremuch smaller than those of the prior art. The large standing wave nodaldistances, as described in the prior art, are much larger than thecut-off diameter that would be useful in the present embodiments. Inaddition, embodiments of the invention described herein include multiplestanding wave regions, which would not be useful in a cell separationdevice.

It should be also noted that unlike nearly every other cell in the body,normal, healthy adipose cells can vary in size over a wide range. Thisis very different from the case often described in the prior art, inwhich different cell types, e.g. erythrocytes and adipocytes, are to beseparated. Erythrocytes, for example, all are within a very narrow sizerange of 6-8 micron. Because they are very small and very dense, thesecells have a high acoustic contrast compared to blood serum and othercells. Thus, they can be separated relatively easily by the prior arttechniques. Those same techniques would be far less effective, if atall, on typical mixtures of adipose cells.

Embodiments of the present invention provide for a new method includingthe lysing of cells above a given size using the operative principle ofgenerating an ultrasonic standing wave field of sufficient intensity andproper geometry, that high shear stress is induced on the cellmembranes. The geometry of the standing wave must be such that thedistance between alternating regions of pressure (particle velocity)fluctuation, i.e. one half the wavelength of the sound wave, is the sameor smaller than the diameter of the cell size to be lysed. The shearstress is generated by having the cell resident in both regions ofalternating pressure (particle velocity), which results in the shearstress being induced upon the cell. This shear stress acts on the cellmembrane to the point of causing cell lysis. Cells smaller than thedistance between the alternating regions of pressure (particle velocity)fluctuation will only be subjected to isotropic compression andrarefaction, which will not induce shear stress, and will not lyse thecell.

The exact ultrasound pressure levels required to cause lysis of thedesired cells depend upon a number of factors, including the viscosityof the surrounding medium, the frequency chosen, the presence of othercells, such as red blood cells, within the medium, the temporal durationof the exposure (dwell time), and is best determined using theguidelines supplied elsewhere herein. In general, the pressure levelswill be on the order of kilopascals to Megapascals. Note, however thatexcessive pressure levels can lead to the onset of cavitation within thefluid, which may act to lyse all the cells within the exposure regionirrespective of size.

To be effective in lysing the cells, sufficient stress must be createdby the ultrasound energy such that it disrupts the cell membrane, ratherthan less intense ultrasound pressure levels that may distort but notinterrupt the membrane surface. Prior art system which disclosedacoustic means for cell separation used limited ultrasound pressurelevels in order to preserve cell integrity.

The small, young, viable cells remaining after the lysing can beseparated from the lysed cells as described herein and then re-insertedinto the patient using techniques commonly used in current medicalprocedures and known to one of ordinary skill in the art.

If stem cells are desired, the size differentiation for lysing is set ata smaller level so that only the smallest, i.e., pre-adipocyte, cellssurvive the lysing process. These cells are further separated from thesuspension to isolate those for stem cell processing.

V. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross sectional representation of a parallel plate standingwave resonator structure.

FIG. 2 is a cross sectional representation of a parallel plate standingwave resonator structure depicting the regions of no particle velocityfluctuation (nodes) and maximal particle velocity fluctuation(anti-nodes).

FIG. 3 is a cross sectional representation of a parallel plate standingwave resonator structure depicting the regions of no particle velocityfluctuation (nodes) and maximal particle velocity fluctuation(anti-nodes), including representations of adipose or other cells of asize less than the distance between nodal and anti-nodal regions.

FIG. 4 is a cross sectional representation of a parallel plate standingwave resonator structure depicting the regions of no particle velocityfluctuation (nodes) and maximal particle velocity fluctuation(anti-nodes), including representations of adipose or other cells of asize as great as or greater than the distance between nodal andanti-nodal regions.

FIG. 5 is an end view representation of a tubular standing waveresonator structure depicting the regions of no particle velocityfluctuation (nodes) and maximal particle velocity fluctuation(anti-nodes).

FIG. 6 is a perspective view of a system for lysing adipocytes thatincludes a cartridge according to an embodiment of the presentinvention.

FIG. 7 is a cross-sectional view of a cartridge that may be used in thesystem shown in FIG. 6.

FIG. 8 is a perspective view of a system for lysing adipocytes thatincludes a number of chambers according to an embodiment of the presentinvention.

VI. DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present invention rely on acoustic standing waves tocreate forces which preferentially affect cells larger than apredetermined, desired size. By controlling the standing wave pattern,the cells which are of a sufficient size to straddle a nodal region(region of minimal pressure or particle velocity) are be affected by thetwo adjacent anti-nodal regions and can be lysed.

In an ultrasonic standing wave field, particles, or in the presentinstance, cells, experience acoustic radiation forces. The primaryradiation force originates from pressure and velocity fluctuations ofthe molecules of the medium. In an ultrasonic standing wave field,acoustic radiation force moves particles toward the pressure nodes orantinodes depending on the acoustic contrast factor “φ,” a function ofdensity and compressibility of the particles and the medium.

The force on a particle (e.g., cell) of radius “r” can be given by:

$F_{PRF} = {{- \left( \frac{2\pi^{2}p_{0}^{2}r^{3}\beta_{m}}{3\lambda} \right)}{\varphi \left( {\beta,\rho} \right)}{\sin \left( {2\; {kx}} \right)}}$

where “p_(o)” is the acoustic pressure amplitude; “λ” the wavelength ofthe acoustic wave, “k” is the wave number; “β_(p)” and “β_(m)” are thecompressibilities of the particle (cell) and medium, respectively,“ρ_(p)” and “ρ_(m)” are the densities of the particle and medium,respectively. The acoustic contrast factor is given by:

${\varphi \left( {\beta,\rho} \right)} = {\frac{{5\rho_{p}} - {2\rho_{m}}}{{2\rho_{p}} + \rho_{m}} - \frac{\beta_{p}}{\beta_{m}}}$

Therefore, particles with different acoustic properties will move todifferent positions (the nodes and antinodes) and can be separated withhelp of a laminar flow in an ultrasound separation device. The goal inembodiments is to exploit the fact that cells which are large comparedto the node to anti-node distance will be subjected to high differentialforces as well. Thus, cells above a certain size can be lysed. Becausethese large cells contain primarily lipid, they are a different densitythan the smaller cells which have less lipid content and proportionallymore internal cellular structures. Thus there will be a differentialforce applied to the different cells. This is in addition to the shearforces that would be induced because the large cells would span thenodal location.

Device 100 shown in FIGS. 1-4 illustrates one embodiment of a device forselectively lysing adipose cells. As used herein, “selectively,”“selectivity,” or “selective” means, in general, that the ultrasoniclysing results in an adipose tissue mass (e.g., liquid with mixture ofcells) more concentrated in small cells than was the original mass. Theexact extent of the selectivity depends on the design of the individuallysing device and the extent and technique of operation as employed on aspecific adipose mass. In each instance, however, the lysing will resultin preferential lysing of cells with a diameter above the predeterminedsize. In some instances, that may result in minimal, if any, lysing ofcells with a diameter below the predetermined size. In other instances,there may be a significant lysing of cells with a diameter below thepredetermined size. But in each instance, lysing is more effective withrespect to cells having a diameter above the predetermined size thanwith a diameter below the predetermined size.

Referring again to device 100, in use, an aqueous solution containing amix of adipose or other cells of a variety of sizes would flow throughthe device 100, between a bottom surface of plate 120 a and a topsurface of plate 120 b. In the embodiment shown in FIG. 1, plates 120 aand 120 b are parallel. With respect to FIG. 2, 3 or 4, the flow wouldbe, for instance, from left to right. With respect to FIG. 5, the flowwould be along the central axis of the cylinder defined by sidewall 120a′. The transducer 110 would generate an acoustic wave that istransmitted through a first surface and would generate an acousticstanding wave field. The acoustic wave may be an ultrasonic wave, whichcreates an ultrasonic standing wave field. As the aqueous solutioncontaining the mix of adipose or other cells passed through the acousticstanding wave field, those cells which were large enough to straddle thenodal planes (e.g., cells 152), as shown in FIG. 4, would be subject tohigh shear forces on their cell membranes, caused by the pressure andparticle velocity gradients from anti-node to anti-node. The shearforces lead to cell damage or cell death. Other cells, smaller thanthose just described (e.g., cells 150), would pass through the acousticstanding wave field relatively unaffected by the pressure and velocitygradients. Small cells in a large cluster would also be unaffectedbecause the shear forces would merely break up the cluster, leaving theindividual cells intact.

Different size cells may be destroyed by changing the geometry andacoustic working frequency of the device. The distance 130 between thebottom surface of plate 120 a and the top surface of 120 b must be anintegral number of wavelengths of the acoustic standing wave field, ascan be appreciated from FIG. 1. The wavelength of the acoustic wave isrelated to the operating frequency by the equation λ=c/f, where “λ” isthe wavelength, “c” is the speed of sound in the aqueous medium, and “f”is the operating frequency. The distance between the bottom surface ofplate 120 a and the top surface of plate 120 b (or in the alternateembodiment, the diameter of the cylinder formed from sidewall 120 a′) isin embodiments an exact integer multiple of the wavelength in order tocreate a standing wave pattern. Further, the distance can be increasedto any reasonable integral number of wavelengths. The larger thedistance, the more volume flow can be achieved, and therefore, thelarger throughput. However, the acoustic waves will be attenuated andabsorbed as they traverse the distance from the bottom surface of plate120 a to the top surface of plate 120 b, so that the destructive effectwill be lessened if the distance is too great.

Another issue to be considered in the design of device 100 for aparticular application is the dwell time, or the time that any specificadipose or other cell would be within the acoustic standing wave field.This clearly depends upon the speed of the cells through the acousticstanding wave field (which is linked to the flow rate), as well as thelongitudinal extent of the standing wave (which is determined by thesize of the transducer 110). The longer the dwell time, the morecomplete the process will be in affecting cells above the desired size.

In some embodiments, the device, may include a series of chambersinterconnected such that the cellular material passes sequentiallythrough them. FIG. 8 illustrates an embodiment 800 with a series ofchambers 802, 804, and 806 each of which is designed to lyse cells ofdifferent diameters. The cells would be transported through the seriesof chambers 802, 804, and 806, starting with one designed to lyse thelargest expected cell size, e.g., 802. Subsequent chambers 804 and 806would be designed for progressively smaller cells. As shown in FIG. 8,each chamber includes a transducer (802A, 804A, and 806A) connected to afirst plate (802B, 804B, and 806B) with top and bottom surfaces, and asecond plate (802C, 804C, and 806C) with top and bottom surfaces. Anacoustic standing wave field is created between the first plates and thesecond plates.

The device in FIG. 8 affords several features. First, the aqueous mediumis slowly cleared of the largest cells, which tend to absorb sound andtherefore can interfere with the acoustic standing wave process if theyare substantially larger than the “cut off” size. By removing thelargest cells (using the lowest frequency), there is less interferencefor subsequent, higher frequency chambers 804 and 806. Second, verylarge cells, or cell clusters, could clog a chamber designed for thesmallest “cut off” size, so it would be advantageous to have theseclusters or large cells disrupted prior to entering the smallestchamber. Additionally, it may be desirable to have multiple outputs fromthe system as shown by the containers 808, 810, and 812, for instance,one output would be a mixture of cells below a given size, the sizeselected to include both adipose cells as well as the smallerpre-adipocytes. A further output may contain only the pre-adipocytes,which are the smallest cells.

As those with skill in the art will appreciate, transducer 110 (or 110′,620, 802A, 804A, and 806A) may be any suitable transducer for generatingan acoustic standing wave field. One non-limiting example is atransducer made from a piezoelectric ceramic material, such as leadzirconate titanate (PZT). The properties of the transducer materials canbe adjusted to suit the application, and in this design, a so-called“hard” ceramic, with a higher mechanical Q is useful in some embodiments(e.g. DoD Type I or PZT-4 from Morgan Electro Ceramics). This approachminimizes the electrical energy needed by minimizing the internalmechanical and dielectric losses within the ceramic material,transferring most of the energy into the acoustic standing wave field.Those skilled in the art of piezoelectric transducers will appreciatethat different materials may be used to suit the necessary design goals,such as cost, electrical characteristics, etc.

Referring again to FIGS. 1-4, the plates 120 a and 120 b must be inintimate contact with the working fluid in order to create and maintainthe acoustic standing wave field. The materials selected for thesesurfaces must provide for easy cleaning and proper acousticalproperties. For example, stainless steel may be used because of its highacoustic reflection coefficient and impermeability and resistance tocorrosion.

In one embodiment, a pump (e.g., pump 612 shown in FIG. 6) or otherforced flow means is used to create the flow of aqueous solutioncontaining the cellular material through the acoustic standing wavefield, rather than using gravity or some other natural flow mechanism.By using a pump, the flow rate can be properly controlled, assuring theproper dwell time for the cellular material within the treatmentchamber(s). Because the dimensions of the treatment chamber mayconstrict the flow, a positive pumping means would generally be requiredin order to overcome the flow resistance presented by the treatmentchamber. In those embodiments that use a pump, it is important that thepumping arrangement be designed so that it does not cause cell lysis,which would negate the desired goal of maintain cell viability for cellsbelow the predetermined size.

The use of an acoustic standing wave field, as used in embodiments ofthe present invention, is very different from filtering orcentrifugation, in that it involves active manipulation of the cellsusing pressure or particle velocity gradients. Filtering causes largecells to be held back in the filter medium while the smaller cells passthrough. However, the filter can become clogged, reducing efficiency.Centrifugation is a batch process which takes additional time tocomplete. Further, after centrifugation, the desired cells must be drawnoff, but there is generally no way to determine where the cells of acertain size are within the centrifugation column.

The approach described herein offers a number of advantages overfiltering or centrifugation. Like the filtering approach, the standingwave method allows for flow-through processing. Unlike filtering, thestanding wave approach permits a continuous flow, with the possibilityof reflow through the device. The centrifugation approach is a batchprocess, which requires additional handling and manipulation of thecells.

The approach described herein allows device embodiments to be adjustedor tuned for the size of the cell to be destroyed by changing thespacing of the node and anti-nodal zones. This is done by a change inthe resonant frequency and the distance between the bottom surface ofplate 120 a and the top surface of plate 120 b. Determining the optimalfrequency for a given desired result requires minimal experimentation.

Those of skill in the art familiar with the issue of cell separation aremostly familiar with the established mechanisms of filtration andcentrifugation. Most of the work currently being described involvesspecific variations on these approaches, such as custom designedfiltering systems with specific pore sizes and ports for sampling andwithdrawing filtered material. Similarly, centrifuges are being designedwith holders that can quickly adapt to fat harvesting and injectionsyringes. None of these approaches involve ultrasound, nor are theydesigned for the continuous flow approach described herein. The use ofultrasound standing waves is unique and novel in this market.

While the drawings depict two embodiments for the device describedherein, one being a parallel plate geometry and the other being acylindrical geometry, those of skill in the art would readily appreciatethat other embodiments are possible. Any geometrical configuration whichgenerates an acoustic standing wave, and which can contain adistribution of cells to be processed would fall within the scope ofthis disclosure. For instance, it is not necessary that the chamberprovide for a “flow through” configuration, and a “batch” processarrangement may be desired for specific applications. In this case, theparallel plate geometry may include a circular transducer geometry, withthe opposing, reflecting plate being the bottom of a cylindricalcontainer. For example, cells in suspension may be placed in acylindrical container, whether tall in aspect ratio (e.g. a graduatedcylinder) or short in aspect ratio (e.g. a Petri dish). A circulartransducer of diameter slightly less than the diameter of the cellcontainer could be lowered into the container until the distance betweenthe transducer and the bottom of the container satisfied the standingwave criteria noted elsewhere. At that distance, the transducer isenergized and the cells are treated. After an appropriate dwell time(determined experimentally for the type of cells to be treated, thetotal volume to be treated, and the available transducer power), thetransducer is turned off and withdrawn, so that there remains a cellsuspension with the cells above a certain dimension lysed. This isclearly a “batch” procedure, but would be suitable for controlledlaboratory experimentation with small cell volumes.

Once the cells have been treated so as to destroy the cells over acertain size, the suspension can be further processed, depending uponthe clinical requirements. The lysed cells will release the lipidmaterial stored within them, so there will be free lipid materialdispersed within the cell suspension. Since the free lipid is not asdense as the intact cells or typically the aqueous solution of the cellsuspension, the lipids will rise to the top of the suspension, wherethey are easily drawn off. Further, the cell remnants (e.g., membranesof disrupted cells that have discharged their contents) are denser thanthe other intact cells (because they no longer contain the lower densitylipid material within them), so again, these remnants will settle to thebottom of the cell suspension. The desired cell material can thus beeasily distinguished from the other, unwanted material within theaqueous suspension, facilitating its collection for re-implantation.

In some embodiments, after lysing large cells, the solution is subjectedto a separation process to separate the smaller intact cells from thecell remnants and other components of the solution. Example separationprocesses may include one or more of filtration, centrifugation, andsettling. These processes may be aided by adding materials, i.e,chemicals, that assist in separating the various components of thesolution. As described above, lysing of the larger cells makes theseparation processes more effective than if the solution were processedwithout lysing. Additionally, since the lysed cells have liberated theirinternal lipid contents, the remnant cellular material is of a muchhigher density and compressibility than intact cells. This makes themmore differentiable with regard to acoustic separation, allowing theprocess to have a secondary utility. Note that in the design of anoverall lysing and separation system, acoustic separation may beaccomplished in a second chamber distinct from the chamber whichproduces cell lysing. A succession of devices, each tuned to a differentcell or cluster size, may be used to improve the overall functioning ofthe system.

In those embodiments in which it is desired to have only stem cells, orpre-adipocytes, the process is essentially the same, save for thedetermination of the cell diameter to be lysed. Stem cells andpre-adipocytes are smaller and denser than adipocytes. Once theadipocytes have been lysed using the method described herein, the stemcells and pre-adipocytes may be removed using the technique justdescribed, especially since these desired cells contain little or nolipid material.

As a potentially beneficial side effect of this method, the remaining(desired) cells will have been stimulated by the action of the acousticstanding wave field. While the force on these cells should be primarilyisotropic, there will be some residual shear stress. Thesemicro-stresses and so-called “micro streaming” around cells can serve to“activate” the cell membranes, allowing for increased biochemicalactivity and thereby a promotion of cell growth. This is usefulregardless of whether the lysing is used to isolate cells forre-insertion or for research or therapeutic use.

In some embodiments, the unlysed cells (adipocytes and stem cells)remaining after lysing are used therapeutically by being reinserted intoa patient. As those with skill in the art will appreciate, these cellscan undergo additional processing to prepare the cells forreintroduction. In some embodiments, after the lysing process, theunlysed cells are separated from other components of the liquid and arefurther processed to stimulate the cells prior to reinsertion into apatient. This further processing may involve a number of biochemicalsteps that improve the cells' ability to be used therapeutically such asby increasing their activity or longevity.

System 600 shown in FIG. 6, illustrates a system for harvestingadipocytes and stem cells from a surgical site 602, and processing asolution containing adipocytes and stem cells. System 600 includes acannula 604 that provides a pathway for removing the adipocytes and stemcells from the surgical site 602. A standing wave resonator device 608is connected to cannula 604 through tubing 606. Tubing 610 connectsstanding wave resonator device 608 to a pump 612 The pump 612 isoperated to create suction at the tip of cannula 604 causing theadipocytes and stem cells to flow from surgical site 602 throughstanding wave resonator device 608 and into a reservoir 616 connected topump 614. The pump 612 is connected to reservoir 616 with tubing 614.The reservoir 616 stores the adipocytes and stem cells for furtherprocessing.

As shown in FIG. 6, system 600 provides for in-stream lysing ofadipocytes that are in a solution of adipocytes and stem cells. Standingwave resonator device 608 may be any suitable resonator for creating astanding wave, such as the resonator devices described above withrespect to FIGS. 1-5. As shown in FIG. 6, device 608 includes aremovable cartridge 618. The solution of stem cells and adipocytes isstored within cartridge 618 which acts as a chamber for holding thesolution during the process of lysing the larger adipocytes.

The walls of cartridge 618 are acoustically transparent, which allowsultrasonic energy to pass through with minimal interference. Also,cartridge 618 is sealed so that outside contaminants will not enter theinterior of the cartridge. In operation, transducer 620 generates anacoustic wave that is transmitted through a first surface of plate 622and generates an acoustic standing wave field between plates 622 and624. As a result of the acoustically transparent nature of cartridge618, the standing wave is created and maintained within the removablecartridge 618. As the solution containing the mix of adipocytes, stemcells, and or fluids passes through removable cartridge 618, those cellswhich are large enough to straddle the nodal planes as shown in FIG. 4,are subject to high shear forces on their cell membranes, caused by thepressure and particle velocity gradients from anti-node to anti-node.The shear forces lead to cell damage or cell death. Other cells wouldpass through the removable cartridge 618 relatively unaffected by thepressure and velocity gradients.

Removable cartridge 618 provides a convenient way to ensure thesterility of the environment during lysing. In some embodiments,cartridge 618 is disposable, so that each time device 608 is used;removable cartridge 618 is replaced with a new cartridge. In otherembodiments, cartridge 618 may be reposable meaning that it must besterilized prior to reuse. However, because removable cartridge 618 isdesigned to be easily removed from device 608, it can easily be removedfrom device 608 and sterilized without being connected to othercomponents of device 608.

As shown in the embodiment of FIG. 6, the solution of adipocytes andstem cells does not directly contact any portion of device 608 exceptfor cartridge 618. The solution flows through tubing 606, cartridge 618,and tubing 610. The other components of device 608 are used to generatethe standing wave but do not come in contact with the solution ofadipocytes and stem cells. For this reason, in some embodiments, tubing606, cartridge 618, and tubing 610 are disposable and used only onetime.

FIG. 7 illustrates a cross-sectional view of an embodiment of acartridge 700 that may be used as cartridge 618 in system 600 (FIG. 6).Cartridge 700 includes walls 702A and 702B, which are acousticallytransparent. That is, they allow sound waves, e.g., ultrasonic soundwaves, to be transmitted through them with little or no interference.This allows a standing wave to be created in the interior of cartridge700. Those with skill in the art will appreciate that acousticallytransparent materials that are suitable for this application includewithout limitation plastics, such as REXOLITE® a cross linkedpolystyrene microwave plastic made by C-Lec Plastics, Inc. Philadelphia,Pa. The particular type of material used may be selected based on theexact frequency of the acoustic wave, the chemical composition of thesolution that includes the adipocytes and stem cells, and otherenvironmental factors such as whether the cartridge is disposable orreposable and will be sterilized in an autoclave.

Cartridge 700 also includes connections 704 and 706 that allowscartridge 700 to be inserted into the flow path of a solution thatincludes adipocytes and stem cells. Connections 704 and 706 may in someembodiments be conventional connections that allow cartridge 700 toconnect to, for example, tubing. The connections 704 and 706 may includethreads, clamps, knobs, or other types of fasteners/connectors. Inaddition to the features that allow cartridge 700 to connect to a flowpath of a solution of adipocytes and stem cells, connectors 704 and 706may also include connectors that allow cartridge 700 to be securedwithin a wave resonator device, such as those devices described above inFIGS. 1-6.

In some embodiments, cartridge 700 also includes an interior coating 708that is applied to the interior walls of cartridge 700. The coating 708may be a membrane, a continuous layer, or a discontinuous layer. Thecoating may include any number of materials that are useful forprocessing the solution of adipocytes and stem cells. For example, thecoating 708 may include chemicals that assist in destroying the cellmembranes of the larger adipocytes during the lysing process. Otherexamples include chemicals that prolong the life of the cells insolution or that assist in breaking up groups of cells. The coating 708may also provide a barrier to contaminants

VII. EXAMPLES Example 1

As a design example, assume that it is desired to preferentially affectcells at or above 250 microns (0.250 mm) in diameter. Thus the distancebetween the anti-nodal planes 142 and 144 would be set to 250 microns.The nodal planes are illustrated as 140. From this, the wavelength ofthe acoustic standing wave field would be 0.5 mm. Assuming a speed ofsound in the aqueous medium of 1500 m/s (similar to that of water), thenthe frequency of operation is 3 MHz. Numerous transducers of differentsizes, dimensions, and power handling capabilities are available at thisfrequency. The distance between the bottom surface of plate 120 a andthe top surface of plate 120 b would then need to be an integral numberof wavelengths, for example 1.0, 1.5, 2.0, 2.5 mm, etc. The distancebetween the plates also affects the total flow through the device 100,as a larger distance allows more fluid to flow through, assuming aconstant transit velocity for the aqueous solution containing the cells.

1-8. (canceled)
 9. An apparatus for lysing cells of greater than orequal to a predetermined diameter in a mixture of cells of a pluralityof diameters, the apparatus comprising: a transducer that vibrates togenerate an acoustic wave; and a first surface connected to thetransducer and positioned at a distance from a second surface, whereinthe first surface transmits the acoustic wave generated by thetransducer to the second surface and creates an acoustic standing wavefield between the first surface and the second surface, the acousticstanding wave field having a wavelength that is no more than twice thepredetermined diameter and the acoustic standing wave field exertingshear forces on exterior membranes of cells with a diameter equal to orgreater than the predetermined diameter.
 10. The apparatus of claim 9,wherein the first surface is a curved surface.
 11. The apparatus ofclaim 10, wherein the first surface is an interior surface of a sidewall of a cylinder.
 12. The apparatus of claim 9, wherein the firstsurface is a flat surface.
 13. The apparatus of claim 12, wherein thefirst surface is a bottom surface of a first plate and the transducer iscoupled to a top surface of the first plate.
 14. The apparatus of claim13, wherein the second surface is flat and is a top surface of a secondplate that is substantially parallel to the first plate.
 15. Theapparatus of claim 9, wherein the second surface is an interior surfaceof a container holding the mixture of cells.
 16. The apparatus of claim9, wherein the distance is an integer multiple of the wavelength. 17.The apparatus of claim 9, wherein the apparatus further comprises: asecond transducer that vibrates to generate a second acoustic wave of asecond wavelength that is less than the first wavelength, wherein thesecond wavelength is no more than twice a second predetermined diameterof adipose cells; and a third surface connected to the second transducerand positioned at a second distance from a fourth surface, wherein thethird surface transmits the acoustic wave generated by the transducer tothe fourth surface and creates a second acoustic standing wave fieldbetween the third surface and the fourth surface, the second acousticstanding wave field exerting shear forces on exterior membranes of cellswith a diameter equal to or greater than the second predetermineddiameter.
 18. The apparatus of claim 9, wherein when a mixture ofadipose cells of a plurality of diameters is subjected to the acousticstanding wave field, adipose cells with diameters equal to or greaterthan the predetermined diameter are selectively lysed compared to cellsof a smaller diameter.
 19. An apparatus for lysing cells of greater thanor equal to a predetermined diameter in a mixture of cells of aplurality of diameters, the apparatus comprising: a transducer thatvibrates to generate an acoustic wave; and a first surface connected tothe transducer and positioned at a distance from a second surface,wherein the first surface transmits the acoustic wave generated by thetransducer to the second surface and creates an acoustic standing wavefield between the first surface and the second surface, the acousticstanding wave field having a wavelength that is no more than twice thepredetermined diameter; a cartridge configured to hold the mixture ofcells, wherein the cartridge is positioned between the first surface andthe second surface and, wherein at least a portion of the standing waveacoustic field is within an interior of the cartridge, the acousticstanding wave field exerting shear forces on exterior membranes of cellswith a diameter equal to or greater than the predetermined diameter. 20.The apparatus of claim 19, wherein the cartridge is disposable.
 21. Theapparatus of claim 19, wherein the cartridge includes walls made ofacoustically transparent material.
 22. The apparatus of claim 21,wherein the acoustically transparent material comprises a plasticmaterial.
 23. The apparatus of claim 19, wherein when the mixture issubjected to the acoustic standing wave field, adipose cells withdiameters equal to or greater than the predetermined diameter areselectively lysed compared to cells of a smaller diameter. 24.-27.(canceled)